Hypoxia-induced cell damage is reduced by mild hypothermia and postconditioning with catalase <em>in-vitro</em>: Application of an enzyme based oxygen deficiency system

Mild hypothermia and pharmacological postconditioning are widespread therapeutical treatment options that positively influence the clinical outcome after tissue hypoxia. In the study presented, a two-enzyme based in-vitro oxygen deficiency model in combination with cultured HT-1080 fibrosarcoma cells was employed to mimic the in-vivo situation of hypoxia and to evaluate the influence of mild hypothermia and postconditioning with catalase on hypoxia-mediated cell damage. Using the in-vitro oxygen deficiency model, partial pressure of oxygen was rapidly reduced to levels below 5mmHg in the culture media and cells responded with an increased expression of hypoxia inducible factor-1 on protein level. Hypoxia resulted in significant cell rounding and retraction of cytoplasmic cell extensions. Evaluation of cytotoxicity revealed a 3.5-fold increase in lactate dehydrogenase levels which was accompanied by 40-fold elevated levels of hydrogen peroxide. The hypoxia-induced increase of lactate dehydrogenase was 2.5-fold reduced in the hypothermia group, although morphological correlates of cytotoxicity were still visible. Hypothermia did not significantly influence hydrogen peroxide concentrations in the culture media. Pharmacological postconditioning with catalase however dose-dependently decreased hypoxia-induced lactate dehydrogenase release. This cytoprotective effect was accompanied by a dose-dependent, up to 50-fold reduction of hydrogen peroxide concentrations and retention of normal cell morphology. We suggest that the described in-vitro oxygen deficiency model is a convenient and simple culture system for the investigation of cellular and subcellular events associated with oxygen deficiency. Moreover, our in-vitro results imply that catalase postconditioning may be a promising approach to attenuate hypoxia-induced and hydrogen peroxide-mediated cell and tissue damage.

轻度低温和药物后处理是广泛应用的治疗选择，对组织缺氧后的临床结果有积极影响。在本研究中，采用基于两种酶的体外缺氧模型结合培养的HT - 1080纤维肉瘤细胞来模拟体内缺氧情况，并评估轻度低温以及过氧化氢酶后处理对缺氧介导的细胞损伤的影响。利用体外缺氧模型，培养基中的氧分压迅速降至5mmHg以下，细胞在蛋白质水平上缺氧诱导因子 - 1的表达增加。缺氧导致细胞明显变圆以及细胞质细胞突起回缩。细胞毒性评估显示乳酸脱氢酶水平增加了3.5倍，同时过氧化氢水平升高了40倍。在低温组中，缺氧诱导的乳酸脱氢酶增加量降低了2.5倍，尽管细胞毒性的形态学相关表现仍然可见。低温对培养基中的过氧化氢浓度没有显著影响。然而，过氧化氢酶的药物后处理呈剂量依赖性地降低了缺氧诱导的乳酸脱氢酶释放。这种细胞保护作用伴随着过氧化氢浓度呈剂量依赖性地降低，最多可降低50倍，并保持正常细胞形态。我们认为，所描述的体外缺氧模型是一种方便、简单的培养系统，可用于研究与缺氧相关的细胞和亚细胞事件。此外，我们的体外研究结果表明，过氧化氢酶后处理可能是一种有前景的方法，可减轻缺氧诱导的以及过氧化氢介导的细胞和组织损伤。